The Regulation Role Of Porcine Prostaglandin Reductase 1(PTGR1) In Inflammation And Oxidative Stress Induced By PRRSV

When the body was exposed to stimulation caused by microbial infections and so on,complex biological responses were aroused such as inflammation and oxidative stress.Severe inflammatory diseases like interstitial pneumonia were induced by porcine reproductive and respiratory syndrome virus(PRRSV),which has caused great losses to the global swine industry.Prostaglandin reductase 1(PTGR1),as a bifunctional enzyme,plays an important role in inflammatory diseases and oxidative stress.However,the role of PTGR1 in pigs infected by PRRSV remains unclear.In this study,pig alveolar macrophages infected by PRRSV were used as a model,and the role of PTGR1 was initially explored in the inflammation and oxidative stress in PRRSV infection.The main results are as follows:(1)Sequence analysis,cloning and antibody preparation of porcine PTGR1 gene:The genetic evolution relationship of porcine PTGR1 was determined and the protein properties and structure were predicted by bioinformatics analysis softwares such as MEGA 7.0.Porcine PTGR1 gene was amplified from the porcine alveolar macrophage cDNA library.A prokaryotic expression vector was constructed,and the His-tagged PTGR1 recombinant protein was expressed and purified,which used to immunize mice and prepared mouse polyclonal antibodies.(2)Anti-inflammatory effect of porcine PTGR1 after PRRSV infection: Pig alveolar macrophages infected by PRRSV were used as a model.At the same time,the expression of porcine PTGR1 during PRRSV infection was first increased and then decreased by q RT-PCR,western blot and flow cytometry.The overexpression of PTGR1 inhibited the expression of proinflammatory cytokines TNF-α,IL-1β and IL-6in porcine alveolar macrophages infected with PRRSV by q RT-PCR and ELISA experiments.And knockdown of PTGR1 would promote the expression of TNF-α,IL-1β and IL-6.These results showed that PTGR1 could suppress inflammation after PRRSV infection to a certain extent.(3)Anti-oxidative stress effect of porcine PTGR1 after PRRSV infection: It was determined that porcine PTGR1 was mainly distributed in the cytoplasm and localized to mitochondria by immunofluorescence assay.The expression of PTGR1 could reduce the expression of reactive oxygen species and the content of malondialdehyde to a certain extent in porcine alveolar macrophages infected with PRRSV by Flow cytometry and other experiments.These results indicated that PTGR1 could suppress oxidative stress to a certain extent.In summary,porcine PTGR1 gene was successfully cloned,and purified recombinant protein was used to prepare mouse polyclonal antibody.The porcine PTGR1 was expressed in the cytoplasm and localized on the mitochondria.The expression of porcine PTGR1 during PRRSV infection was first increased and then decreased.At the same time,it was preliminarily determined that porcine PTGR1 had anti-inflammation and anti-oxidative stress effects on PRRSV infection to a certain extent,which was laid the foundation of later research on its anti-inflammation and anti-oxidative stress mechanisms and provided a theoretical basis for the treatment of inflammatory diseases caused by PRRSV.